Ultrastructural effect of sildenafil citrate on corpus cavernosum and other genital organs in female rats

Asian J Androl 2003 Mar; 5: 37-41             

Keywords: sildenafil; female; clitoris; histopathologic effects

Abstract

Aim: To determine the ultrastructural effects of sildenafil on the female genital organs. Methods: Twenty female cycling Wistar albino rats weighing 250 20 g were randomly divided into two groups of 10 each. Rats of one group were gavaged with 0.5 mg.kg^-1.d^-1 of sildenafil 3 days in a week for 4 weeks and the other served as the controls. After cessation of treatment animals were sacrificed by cervical dislocation under methoxyflurane anaesthesia. The clitoris, vagina, uterus and bartholin glands were taken at the estrous and were fixed with 10 % formalin solution for light microscopy and 2.5 % glutaraldehyde and osmic acid for electron microscopy. Results: Under the light microscope, the fibrocollageous tissue was found increased, the capillaries enlarged and the connecting tissue elements increased in the corpus cavernosum in the treated group. On electron microscopy, increased connective tissue, fibroblasts with notched nucleus, shorten immature collagen fibers without striation were seen. Abundant foldings and penetration with collagen bundles were observed in the basal membrane. Large connection complexes, especially gap junctions among the wide capillary endothelial cells were observed. Conclusion: There are evident histological changes due to sildenafil citrate in female rat corpus cavernosum. The clitoris and bartholin glands were the most effected organs. While the histopathological changes of clitoral tissue could be expected, an increase in the mass of bartholin gland was surprised.

1 Introduction

Sildenafil citrate has been used widely in the treatment of erectile dysfunction (ED) with various etiologies. The drug, a pyrazolo-pyrimidinyl-methylpiperazine compound, is a selective inhibitor of the cyclic guanosine monophosphate (cGMP)- specific phosphodiesterase type 5 (PDE5) [1, 2]. It enhances the relaxation effect of nitric oxide (NO) on the corpus cavernosum by inhibiting the PDE5, which is responsible for the degradation of cGMP [3]. The inhibition of PDE5 by sildenafil citrate increases the cGMP levels in the corpora, causing smooth muscle relaxation and blood flow increase into the penis, whenever there is local release of NO by sexual stimulation. This is the main action of sildenafil on erection [3, 4].

Nowadays, there are many publications about the beneficial effects of sildenafil on women with sexual dysfunction, but its long term ultrastructural effects on the target tissues, especially the female genital organs, are not well understood. The present study was designed to investigate the histopathological effects of sildenafil on the clitoral corpus cavernosum and other genital organs in female rats by light and electron microscopy.

2 Materials and methods

Twenty female cycling Wistar albino rats weighing 250?0 g were used and housed in standard pathogen free (SPF) conditions. They were randomly divided into two groups of 10 animals each. Rats of one group were gavaged 0.5 mg.kg-1.d-1 of sildenafil 3 days in a week for 4 weeks as suggested by Ishikura F, et al. (Am Urol Ass 95th Annual Meeting, Atlanta- Georgia, J Urol 2000 Abstract No. 887) and the other served as the controls. The dose is less than that for the humans on per kg body weight basis and is much less than the conventional dose for rats. After cessation of treatment, vaginal smears were obtained from animals at estrous when the vaginal orifice was dilated and had lighter colour as compared with those at proestrous. A drop of water was placed into the vagina and was aspirated several times. The aspirate was transferred onto a slide and made a smear. It was dried, soaked for 5 seconds in absolute methyl alcohol and again dried in air at room temperature. The slide was placed in 1/20 Giemsa solution for 30 minutes and then rinsed in water. It was observed under microscope after dryness. Flat and irregular large cornified epithelial cells were observed at estrous. Animals at estrous were sacrificed by cervical dislocation under methoxyflurane anaesthesia. The clitoris, vagina, uterus and bartholin glands were dissected and fixed in 10 % formalin for light microscopy and in 2.5 % glutaraldehyde and osmic acid for electron microscopy. The slides were evaluated by 3 histologists on a blind basis.

Slides were washed overnight with tap water and were then dehydrated through a graded series of ethanol (50 %, 60 %, 70 %, 85 %, 95 % and 100 %), 30 minutes for each step. They were incubated in a 50:50 mixture of 100 % ethanol and xylene for 30 minutes and followed by two washes in xylene for 1 h. After that, they were transferred to xylene and paraffin embedding mixture for 30 minutes. Transverse sections (5 µm) were taken and dewaxed under 60 overnight. Sections were immersed in xylene for 1 h and rehydrated through a graded series of ethanol (100 %, 95 %, 80 %, 70 % and 60 %) for 2 minutes in each concentration and were then washed in tap water. They were stained with haematoxylin and eosine (H-E). Slides were mounted using entellan and covered with coverslips prior to viewing and photography under the Olypmpus BX-40 light microscope.

After fixation, the specimens were stained with uranyl acetate and processed through standard dehydration in graded ethanol before infiltration and embedded in Epon. The semi-thin sections were stained with toluidine blue-azur II before examined under an electron microscope; ultrathin sections were cut on a Leica Ultracut R ultramicrotom and stained with uranyl acetate-lead citrate before viewing with a Zeiss 95 M electron microscope.

3 Results

Under the light microscope, no clitoral epithelial changes were observed, while the surrounding fibro-collageous tissue was found increased in the treated group; capillaries were enlarged and connecting tissue elements were increased in the corpus cavernosum (Figure 1).

Figure 1. Semi-thin section of clitoris of treated group (100).

Under the electron microscope in the treated group, the semi-thin sections indicated that the number of elements and connecting tissue fibers of the corpus cavernosum were increased; in the ultrathin sections, increased connective tissue and fibroblasts with notched nucleus and shortened immature collagen fibers without striations were observed. These fibroblasts with euchromatic nuclei were accepted as active. Newly synthesized collagen fibers were also found in the connective tissue (Figure 2a). In semi-thin sections of the controls, collagen fibers of the connective tissue were observed less in amount and fibroblasts were not activated (Figure 2b).

Figure 2a. Active fibroblasts and increased collagen fibers in corpus cavernosum of treated group (EM 10000, F: fibroblast, C: collagen, asterisk: newly synthesized dense collagen fibers).
Figure 2b. Fibroblasts and collagen fibers in corpus cavernosum of control group (EM 10000, F: fibroblast; C: collagen fibers).

In semi-thin sections of the endothelial capillaries of corpus cavernosum, as expected, no difference was observed, but in ultrathin sections, in contrary to the stable cells of the control group, cells were active and the nuclei were granulated. The basal membrane surrounding the wide capillary endothelial cells in the corpus cavernosum was seen as a straight line just under the endothelial cell membrane. In addition, the foldings of basal membrane were exaggerated in some areas. The collagen bundles were penetrating the basal membrane of the corpus cavernosum under the endothelial cells in several microscopic fields (Figure 3).

Figure 3. Large endothelial cell and basal membrane in corpus cavernosum of (A) treated group; (B) control group (EM 7250, E: endothelial cells; BM: basal membrane).

There was no significant difference in the small capillary endothelium in both the control and treated groups, while the large capillary endothelial changes were observed in the treated group (Figure 4).

Figure 4: Endothel and basal membrane of the small capillaries in corpus cavernozum of (A) treated group; (B) control group (EM 6250, CL: capillary lumen, BM: basal membrane, E: endothel) .

The connection complexes, especially the gap junctions, among the wide capillary endothelial cells were widened in the treated group (Figure 5).

Figure 5. Connection complexes between endothelial cells of small capillary in corpus cavernosum of (A) treated group; (B)control group (EM 10000, GJ: gap junctions).

In light microscopic examination, the stratified squamous epithelium of vagina was found to be shed in the treated group, while it was normal in the control group.

Additionally, both the connective tissue and the cellular elements of the stroma beneath the epithelium were increased in the treated animals; the connective tissue was compact and dense.

In electron microscopic analysis, semi-thin sections of the vagina revealed a similar picture in regard to the epithelium; increased connective tissue due to collagen fibers was observed as seen in the clitoris. Active fibroblasts were also found in vaginal sections similar to clitoral sections.

The gland was macroscopically enlarged in the treated group. Under the light microscope, columnar cells with mucus secreting pale cytoplasm and small basal nuclei were observed in the treated group with a hyperplasia of the cytoplasmic secreting granules of the gland cells. Young and activated fibroblasts were increased and newly synthesized collagen fibers in the stroma of clitoris were observed (Figure 6).

Figure 6. Semi-thin section of bartholin gland in (A) treated group ; (B)control group (400).

There were no significant changes in the uterus in the treated group as compared with the controls (Figure 7).

Figure 7. Semi-thin section of uterus in (A) treated group; (B) control group (200).

4 Discussion

During the last 10 years, there has been a great development in regard to the pathophysiology and treatment of ED. Unfortunately, only a few studies have been focused on the sexual dysfunction of women. It was pointed out that 18 %~76 % of women complained of sexual dysfunction [5].

Recently, it has been shown that women with sexual dysfunction can be treated with vasodilators [6-10]. Some researchers pointed out that both the clitoral and vaginal blood flow were partially regulated by NO [6, 8, 9, 11, 12]. Further studies should be done to assess the role of cGMP-specific PDE5 in clitoris and vagina. It is suggested that sildenafil citrate used in the treatment of ED could also be an important candidate for the treatment of women sexual dysfunction in the future.

Recent reports pointed out that sildenafil citrate could be used safely in sexual dysfunction of post menopausal women [13]. For this reason, we investigated the histopathological effects of sildenafil on the genital organs of female rat.

Enlargement of capillary in clitoris was observed in our study. This enlargement will undoubtedly result in an increase in blood flow in the clitoral tissue. To overcome the difficulty of clitoreal arousal as a result of atherosclerosis in the postmenopausal period, this affirmative effect of sildenafil on tissue level may be helpful.

Aging and atherosclerotic vascular diseases decreased the ratio of clitoral cavernosal smooth muscle/connective tissue [6], but the effect of clitoral fibrosis on women sexual dysfunction was not clear. In our study, increased cavernosal smooth muscle and surrounding fibrocol-lagenous tissue have been observed. In addition, connective tissue fibers, especially the young and activated fibroblasts, were increased. As a result, plenty newly synthesized collagen fibers were seen in the stroma of clitoris.

Sildenafil citrate increased the vaginal lubrication feeling in women with sexual dysfunction [6]. We observed that in the treated group the connecting tissue elements, the cellular elements in the stroma and the vaginal capillaries beneath the epithelium were increased. These findings support the view that the improvement of lubrication can be due to the increased blood flow and number of stromal elements in the vagina.

An increase in connective tissue and dense collagen fibers in corpus cavernosum was demonstrated. Fibroblastic activation and increase of dense collagen fibers could be irreversible, so prolonged using of sildenafil citrate may result in fibrosis in both clitoral and penile tissues. Sildenafil citrate could improve erectile function by widening the gap junctions, as indicated in the present study.

It can be seen that the clitoris and Bartholin glands were the organs most affected by sildenafil. The clitoral changes could be expected, but an increase in the mass of Bartholin gland somewhat bewildered us.

References

[1] Jeremy JY, Ballard SA, Naylor AM, Miller MA, Angelini GD. Effects of sildenafil, a type-5 cGMP phosphodiesterase inhibitor, and papaverine on cyclic GMP and cyclic AMP levels in the rabbit corpus cavernosum in vitro. Br J Urol 1997; 79: 958-63.
[2] Balard SA, Gingell CJ, Tang K, Turner LA, Price ME, Naylor AM. Effects of sildenafil on the relaxation of human corpus cavernosum tissue in vitro and on the activities of cyclic nucleotide phosphodiesterase isozymes. J Urol 1998; 159:2164-71.
[3] Medina P, Segarra G, Vila JM, Domenech C, Martinez-Leon JB, Lluch S. Effects of sildenafil on human penile blood vessels. Urology 2000; 56: 539-43.
[4] Moreira SG, Brannigan Jr RE, Spitz A, Orejuela FJ, Lipshultz LI, Kim ED. Side-effect profile of sildenafil citrate (Viagra) in clinical practice. Urology 2000; 56: 474-6.
[5] World Health Organisation. The ICD-10 Classification of mental and behavioral disorders: Clinical Descritions an Diagnostic Guidelines. EHO: Genova; 1992.
[6] Berman JR, Goldstein I. Female Sexuel Dysfunction. Urology Clinics of North America 2001; 28/2: 405-16.
[7] Shenn WW, Urosevich Z, Clayton DO. Sildenafil in the treatment of female sexual dysfunction induced by selective serotonin reuptake inhibitors. J Reprod Med 1999; 44: 535-42.
[8] Rajfer J, Aronson WJ, Bush PA, Dorey FJ, Ignarro LJ. Nitric oxide as a mediator of relaxation of the corpus cavernosum in response to nonadrenergic noncholinergic neurotransmission. N Engl J Med 1992 Jan 9; 326: 90-4.
[9] Burnett AL. Nitric oxide in the penis: physiology and pathology. J Urol 1997; 157: 320-4.
[10] Wagner G, Saenz de Tejada I. Update on male erectile dysfunction. BMJ 1998;316: 678-82.
[11] Andersson KE, Wagner G. Physiology of penil erection. Physiol Rev 1995; 75: 191-236.
[12] Medina P, Segarra G, Vila JM, Domenech C, Martinez-Leon JB, Lluch S. Effects of sildenafil on human penile blood vessels. Urology 2000; 56: 539-43.
[13] Kaplan SA, Reis RB, Kohn IJ, Ikeguchi EF, Laor E, Te AE, et al. Safety and efficacy of sildenafil in postmenopausal women with sexual dysfunction. Urology 1999; 53: 481.

home

Correspondence to: Dr. M. Ilker Gndz, Department of Urology, Medical Faculty, Celal Bayar University, 45010 Manisa, Turkey.
Tel: +90-236-232 3133 Fax: +90-236-237 0213
E-mail: ilker_gunduz@hotmail.com
Received 2002-06-27      Accepted 2002-12-17